The present invention relates to a rat ganglioside GM1-specific xcex11xe2x86x922fucosyltransferase. The invention provides novel nucleotide sequences of a rat xcex11xe2x86x922fucosyltransferase specific for a carbohydrate moiety found in ganglioside GM1, more particularly, specific for a terminal galactose xcex21xe2x86x923N-acetylgalactosamine (Galxcex21xe2x86x923GalNAc) saccharide, amino acid sequences of its encoded protein (including peptide or polypeptide), and derivatives and analogs thereof. Merely for the ease of description, the enzyme is herein referred to as xe2x80x9cGM1-specificxe2x80x9d or xe2x80x9cganglioside GM1-specificxe2x80x9d. The invention also relates to fragments (and derivatives and analogs thereof) which comprise a domain of rat ganglioside GM1-specific xcex11xe2x86x922fucosyltransferase with catalytic activity. Methods of production of rat ganglioside GM1-specific xcex11xe2x86x922fucosyltransferase and derivatives and analogs thereof (e.g. by recombinant means) are provided. In addition, the invention relates to methods of inhibiting the function of rat ganglioside GM1-specific xcex11xe2x86x922fucosyltransferase (e.g. by means of antisense RNA). The invention further relates to use of rat ganglioside GM1-specific xcex11xe2x86x922fucosyltransferase in the preparative production of fucosyl-GM1. Applications of fucosyl-GM1, for example as an immunotherapeutic for cancer, are disclosed.
Citation of a reference herein shall not be construed as an admission that such reference is prior art to the present invention.
2.1. Fucosyltransferases
Fucosyltransferases are enzymes that catalyze the addition of a fucose residue to a terminal galactose acceptor of saccharide precursors. Fucosyltransferase activity is involved in the production of oligosaccharides, glycolipids or glycoproteins. There are four known classes of fucosyltransferases, namely those that catalyze the addition of fucose in xcex11xe2x86x922, xcex11xe2x86x923, xcex11xe2x86x924 and xcex11xe2x86x926 linkages.
Fucosyltransferases are best known for their roles in the synthesis of the oligosaccharide moieties that comprise blood group antigenic determinants. For example, the fucosyltransferase encoded by the H gene catalyzes the transfer of fucose in an xcex11xe2x86x922 linkage to the terminal galactose of Gal(xcex21xe2x86x924)GlcNAc(xcex21-3)Gal-R to produce xe2x80x98H substancexe2x80x99 on the surface of erythrocytes. Further addition of N-acetylgalactosamine or galactose leads to the formation of the type A or type B blood group substances respectively. An analogous enzyme encoded by the Se locus catalyzes the formation of xe2x80x98H substancexe2x80x99 in epithelial tissues for secretion rather than presentation at the cell surface (Rosen et al., 1989, Dictionary of Immunology, Stockton Press, New York, pp. 1-3).
Previous experiments with H35 hepatoma cell extracts demonstrated that transfer of fucose to neolacto-series acceptors occurred at a rate only 2% of that found for GM1 (Holmes, E. H., et al, 1983, J. Biol. Chem, 258:3706-3713). This substrate specificity is more restricted compared to other cloned xcex11xe2x86x922fucosyltransferases but is most closely related to secretor-type enzymes (Larsen, R. D., et al., 1990, Proc. Natl. Acad. Sci. USA 87:6674-6678; Kelly, R. J., et al., 1995, J. Biol. Chem. 270:4640-4649; Hitoshi, S., et al., 1995, J. Biol. Chem. 270:8844-8850; Hitoshi, S., et al., 1996, J. Biol. Chem. 271:16975-16981).
2.2. Structure of xcex11xe2x86x922Fucosyltransferases
To date, a number of genes encoding H-type and Se-type xcex11xe2x86x922fucosyltransferases have been cloned from several species of organisms. Three human xcex11xe2x86x922fucosyltransferases (Larsen et al., 1990, Biochemistry 87:6674-6678; Koda et al., 1997, Eur. J. Biochem. 246:750-755; Kelly et al., 1995, J. Biol. Chem. 270:4640-4649), three rabbit xcex11xe2x86x922fucosyltransferases (known as RFT-I, RFT-II and RFT-III) (Hitoshi et al., 1995, J. Biol. Chem. 270:8844-8850; Hitoshi et al., 1996, J. Biol. Chem. 271:16975-19681), and two mouse xcex11xe2x86x922fucosyltransferases (Tsuji, 1996, GenBank accession no. Y09882; Lin et al., 1998, GenBank accession no. AF064792) have been described. Piau et al. (1994, Eur. J. Biochem. 300:623-626) disclose fragments, designated FTA and FTB, of two rat xcex11xe2x86x922fucosyltransferases isolated from rat PROb colon adenocarcinoma cells. Piau et al. showed that antisense expression of the FTA or FTB nucleic acid fragments inhibited the endogenous xcex11xe2x86x922fucosyltransferase activity of PROb cells with respect to the synthetic fucose acceptor phenyl xcex2-D-galactopyranoside; however the FTB fragment was not shown to be sufficient for xcex11xe2x86x922fucosyltransferase catalytic activity, nor was the substrate specificity of the PROb xcex11 2fucosyltransferase activity determined.
H-type xcex11xe2x86x922fucosyltransferases are membrane localized whereas Se-type xcex11xe2x86x922fucosyltransferases are localized to the Golgi apparatus. Amino acid sequence alignment of membrane bound H-type xcex11xe2x86x922fucosyltransferases reveals that, like other glycosyltransferases, there exists a homologous domain structure comprising a short intracellular N-terminal domain, a transmembrane domain, an extracellular stem region not required for enzymatic activity, and finally, the catalytic domain at the C-terminus. Generally, there is little sequence homology outside the catalytic domain.
2.3. Ganglioside GM1 and its Fucosylated Derivative Fucosyl-GM1 
Gangliosides are cell surface constituents comprising glycosphingolipids (produced by the linking of ceramides to oligosaccharides) with sialic acid residues. Depending on the number of sialic acid residues they possess, gangliosides are known as mono-, di-, tri- or polysialogangliosides. GM1 stands for ganglioside mono(sialic acid)1.
Fucosyl-GM1, detected by monoclonal antibodies, is found largely in the nervous system, and in particular on a subpopulation of neurons in the dorsal root ganglia and dorsal horn of the spinal cord, as well as on surrounding satellite cells surrounding the fucosyl-GM1 positive neurons (Kusunoki et al., 1989, Brain Res. 494:391-395; Kusonoki et al., 1992, Neurosci. Res. 15: 74-80).
Gangliosides have long been implicated in diseased states. They are often prominent cell surface constituents of transformed cells (see Section 2.5, infra) and alterations in their metabolism give rise to diseases of the nervous system. For example, several fatal hereditary diseases are caused by lysosomal storage of gangliosides wherein the absence or deficiency of lysosomal enzymes results in the deleterious accumulation of gangliosides. The most well known of these diseases is the neurodegenerative Tay-Sachs disease, which is characterized by the accumulation of ganglioside GM2. Accumulation of GM1 results in GM1 Gangliosidosis.
2.4. Regulation of Fucosyltransferase Expression
xe2x80x98H substancexe2x80x99, the fucosylated precursor of blood group determinants, is strictly regulated temporally and spatially during vertebrate development (Fenderson et al., 1986, Dev. Biol. 114:12-21).
Dramatic changes in the expression of cell surface glycolipids are found with oncogenesis (Hakomori, 1989, Adv. Cancer Res. 52:257-331; Alhadeff, 1989, CRC Crit. Rev. Oncol./Hematol. 9:37-107). These changes frequently are oncofetal in nature in that a particular carbohydrate structure may be expressed during normal fetal development, disappear in adult tissues, and reappear in association with oncogenesis giving rise to a premalignant or malignant marker. One such example is expression of the ganglio-B determinant (II3NeuAcIV3 xcex1GalIV2FucGg4) during early stages of chemical carcinogenesis in rat liver with N-2-acetylaminofluorene (AAF) (Holmes and Hakomori, 1982, J. Biol. Chem. 257:7698-7703; Scribner et al., 1983, Environ. Health Perspect. 49:81-89). Expression of this determinant has been shown to be a property of liver parenchymal cells resulting from a carcinogenic stimulus but not hepatotoxicity (Holmes, 1990, Carcinogenesis 11:89-94). This determinant has also been shown to be developmentally regulated in rat stomach (Bonhours et al., 1987, J. Biol. Chem. 258:3706-3713). Expression of this antigen is due to the activation of an xcex11xe2x86x922fucosyltransferase which is normally unexpressed in adult rat liver parenchymal cells. This enzyme efficiently transfers fucose onto the terminal galactose residue of a GM1 precursor, producing fucosyl-GM1 (IV3NeuAcIV2FucGgOse4Cer). Fucosyl-GM1 is a substrate for a constituitively expressed xcex11xe2x86x923galactosyltransferase forming the blood group B determinant on a ganglioside core chain (Holmes and Hakomori, 1983, J. Biol. Chem. 258:3706-3713; Holmes and Hakomori, 1987, J. Biochem. 258:3706-3713). This xcex11xe2x86x923galactosyltransferase behaves as a blood group B transferase in that it efficiently catalyzes transfer of galactose in xcex11xe2x86x923-linkage to terminal galactose residues of xcex11xe2x86x922fucosylated neolacto- and ganglio-series acceptors (Holmes and Hakomori, 1983, J. Biol. Chem. 258:3706-3713).
High xcex11xe2x86x922fucosyltransferase expression is observed in rat hepatoma H35 cells (Holmes and Hakomori, 1983, J. Biol. Chem. 258:3706-3713; Holmes and Hakomori, 1987, J. Biochem. 258:3706-3713). The enzyme from H35 cells has specificity for a ganglio-series core chain. These cells accumulate large amounts of fucosyl-GM1 (Baumann, H., et al., 1979, Cancer Res. 39:2637-2643). Enzymological studies indicated this enzyme was inhibited by a wide variety of detergents, an unusual property for a membrane bound glycosyltransferase (Holmes, E. H., et al, 1983, J. Biol. Chem, 258:3706-3713). This property may reflect a role for membrane phospholipids in maintaining the enzyme in an active conformation (Holmes and Hakomori, 1987, J. Biochem. 101:1095-1105). Later studies demonstrated that active enzyme could be solubilized from H35 cell membranes by 0.4% CHAPSO which bound to the affinity resin GDP-hexanolamine-Sepharose (Holmes, E. H., et al., 1987, J. Biochem. 101:1095-1105).
Further, the observation about the production by transformed cells of high levels of fucosyl-GM1 as a result of xcex11xe2x86x922fucosyltransferase activity, is not restricted to rat hepatoma cells. For example, in humans, fucosyl-GM1 is associated with small cell lung carcinoma (Fredman et al., 1986, Biochim. Biophys. Acta 875:316-323; Nilsson et al., 1984, Glycoconjugate J. 1:43-49).
Generally, enzymatic oligosaccharide synthesis (including synthesis of glycolipids, glycoproteins, etc.) has been limited by the difficulty of isolation and enrichment of glycosyltransferases from natural sources. Thus, there is a need for methods to produce easily isolatable quantities of glycosyltranferases with high enzymatic activity. Such glycosyltransferases, produced, e.g. in vitro, would be useful reagents in compensating for the lack of natural resources. In particular, there is a need for methods to produce easily isolatable GM1-specific xcex11xe2x86x922fucosyltransferase. The ability to synthesize fucosyl-GM1 in vitro is of particularly high value, as the ganglioside is important for the development of the mammalian nervous system. GM1-specific xcex11xe2x86x922fucosyltransferase can be used to catalyze the addition of fucose residues to terminal Galxcex21xe2x86x923GalNAc saccharide chains of glycoproteins, glycolipids, glycolipoproteins and oligosaccharides, producing saccharide compositions that are useful nutritional additives or bases therefor. Further, fucosyl-GM1 is envisaged to be an important tool in cancer therapy and cancer diagnostics. Until the cloning and characterization of the nucleic acid and amino acid sequences of the catalytic domain and the fill length xcex11xe2x86x922fucosyltransferase of the present invention, no xcex11xe2x86x922fucosyltransferases with GM1 specificity had been identified.
The present invention provides a rat ganglioside GM1-specific xcex11xe2x86x922fucosyltransferase. As indicated above, the novel nucleic acids of the invention encode an xcex11xe2x86x922fucosyltransferase enzyme specific for a terminal Galxcex21xe2x86x923GalNAc saccharide found naturally in ganglioside GM1. According to the present invention, the novel nucleic acids encode an xcex11xe2x86x922fucosyltransferase enzyme specific for the terminal Galxcex21xe2x86x923GalNAc moiety which can be a part of a glycoprotein, a glycolipid, a glycolipoprotein or free oligosaccharide or polysaccharide molecule. Merely for ease of description, and not limitation, the enzyme is referred to herein as xe2x80x9cGM1-specificxe2x80x9d or xe2x80x9cganglioside GM1-specificxe2x80x9d. More particularly, the invention encompasses nucleotide sequences of a rat ganglioside GM1-specific xcex11xe2x86x922fucosyltransferase, amino acid sequences of its encoded protein (including peptide or polypeptide), and derivatives and analogs thereof. The invention further encompasses fragments (and derivatives and thereof) which comprise a domain of rat ganglioside GM1-specific xcex11xe2x86x922fucosyltransferase with catalytic activity. Methods of production of rat ganglioside GM1-specific xcex11xe2x86x922fucosyltransferase (e.g. by recombinant means), and derivatives and thereof, are provided. Methods of inhibiting the function of ganglioside GM1-specific xcex11xe2x86x922fucosyltransferase (e.g. by means of antisense RNA) are provided. The invention further encompasses methods for the use of rat ganglioside GM1-specific xcex11xe2x86x922fucosyltransferase in the production of glycoproteins, glycolipids, glycolipoproteins and free oligo- or polysaccharides. Examples of uses of these products, such as uses as nutritional additives, are provided. The methods are particularly useful as they can be used in preparative biosynthesis of these saccharide-containing compositions, and are adaptable to such synthesis in large or commercial scale production. Of particular importance is the synthesis of fucosyl-GM1, which is useful as an immunotherapeutic against cancer and neurological disease.
This invention provides an isolated or purified protein comprising an amino acid sequence as depicted in FIG. 5 (SEQ ID NO:8). The invention further provides an isolated or purified protein comprising amino acids 28-380 of SEQ ID NO:8 as depicted in FIG. 3A (SEQ ID NO:10).
This invention provides an isolated or purified protein consisting of an amino acid sequence as depicted in FIG. 5 (SEQ ID NO:8).
The invention further provides an isolated or purified protein consisting of amino acids sequence numbers 28-380 of SEQ ID NO:8 as depicted in FIG. 3A (SEQ ID NO:10).
This invention provides an isolated or purified protein, the amino acid sequence of which consists of a catalytic domain defined by amino acid numbers 1-353 as depicted in FIG. 3A (SEQ ID NO: 10) or amino acid numbers 28-380 as depicted in FIG. 5 (SEQ ID NO:8).
This invention provides an isolated or purified protein, the amino acid sequence of which consists of amino acid numbers 1-380 as depicted in FIG. 5 (SEQ ID NO:8) covalently linked to at least a portion of a second protein, which second protein is not said protein defined by the amino acid sequences as depicted in FIG. 5 (SEQ ID NO:8). In another embodiment, the protein is fused by a covalent bond to at least a portion of a second protein, wherein said portion is the IgG binding domain of protein A.
This invention provides an isolated or purified protein, the amino acid sequence of which consists of amino acids numbers 28-380 as depicted in FIG. 5 (SEQ ID NO:8) or amino acids numbers 1-353 as depicted in FIG. 3A (SEQ ID NO:10) covalently linked to at least a portion of a second protein, which second protein is not said protein defined by the amino acid sequences as depicted in FIG. 5(SEQ ID NO:8). In another embodiment, the protein is fused by a covalent bond to at least a portion of a second protein, wherein said portion is the IgG binding domain of protein A.
This invention provides an isolated nucleic acid comprising a nucleotide sequence as depicted in FIG. 5 (SEQ ID NO:7).
This invention provides an isolated nucleic acid comprising a nucleotide sequence encoding an amino acid sequence as depicted in FIG. 5 (SEQ ID NO:8).
This invention provides an isolated nucleic acid comprising a nucleotide sequence as depicted in FIG. 3A (SEQ ID NO:9).
This invention provides an isolated nucleic acid comprising a nucleotide sequence encoding an amino acid sequence as depicted in FIG. 3A (SEQ ID NO:10).
This invention provides an isolated RNA molecule comprising a nucleotide sequence as depicted in FIG. 5 (SEQ ID NO:7), wherein the base U(uracil) is substituted for the base T (thymine) of said sequence.
This invention provides an isolated RNA molecule comprising a nucleotide sequence encoding an amino acid sequence as depicted in FIG. 5 (SEQ ID NO:8).
This invention provides an isolated RNA molecule comprising a nucleotide sequence as depicted in FIG. 3A (SEQ ID NO:9), wherein the base U(uracil) is substituted for the base T (thymine) of said sequence.
This invention provides an isolated RNA molecule comprising a nucleotide sequence encoding an amino acid sequence as depicted in FIG. 3A (SEQ ID NO:10).
This invention provides an isolated nucleic acid comprising a nucleotide sequence that is the reverse complement of a nucleotide sequence encoding an amino acid sequence as depicted in FIG. 5 (SEQ ID NO:8).
This invention provides an isolated nucleic acid comprising a nucleotide sequence that is the reverse complement of a nucleotide sequence encoding an amino acid sequence as depicted in FIG. 3A (SEQ ID NO:10).
This invention provides a vector comprising (a) a nucleotide sequence as depicted in FIG. 5 (SEQ ID NO:7)and (b) an origin of replication. In one embodiment, the nucleotide sequence is operably linked to a heterologous promoter.
This invention provides a vector comprising (a) a nucleotide sequence as depicted in FIG. 3A (SEQ ID NO:9)and (b) an origin of replication. In one embodiment, the nucleotide sequence is operably linked to a heterologous promoter.
This invention provides a vector comprising (a) a nucleotide sequence that is the reverse complement to all or a fragment of the nucleotide sequence as depicted in FIG. 5 (SEQ ID NO:7) and (b) an origin of replication. In one embodiment, the nucleotide sequence is operably linked to a heterologous promoter.
This invention provides a vector comprising (a) a nucleotide sequence that is the reverse complement to all or a fragment of the nucleotide sequence as depicted in FIG. 3A (SEQ ID NO:9) and (b) an origin of replication. In one embodiment, the nucleotide sequence is operably linked to a heterologous promoter.
The invention provides a vector comprising (a) a nucleotide sequence encoding an amino acid sequence as depicted in FIG. 5 (SEQ ID NO:8) and (b) an origin of replication.
The invention provides a vector comprising (a) a nucleotide sequence encoding an amino acid sequence as depicted in FIG. 3A (SEQ ID NO:10) and (b) an origin of replication.
The invention provides a recombinant cell containing a recombinant nucleic acid vector comprising a nucleotide sequence as depicted in FIG. 5 (SEQ ID NO:7). In one embodiment, the recombinant cell is a eukaryotic cell and preferably a mammalian cell.
The invention provides a recombinant cell containing a recombinant nucleic acid vector comprising a nucleotide sequence as depicted in FIG. 3A (SEQ ID NO:9). In one embodiment, the recombinant cell is a prokaryotic cell and preferably a bacterial cell.
This invention provides a method of producing a rat xcex11xe2x86x922fucosyltransferase protein comprising: (a) culturing a recombinant cell containing a vector comprising a recombinant nucleotide sequence as depicted in FIG. 5 (SEQ ID NO:7), such that the xcex11xe2x86x922fucosyltransferase protein, encoded by SEQ ID NO:7, is expressed by the cell; and (b) recovering the expressed protein or a cellular fraction containing said protein. In one embodiment, the invention provides the purified protein produced by the method. In another embodiment, the invention provides a cellular fraction with said protein activity.
This invention provides a method of producing a rat xcex11xe2x86x922fucosyltransferase protein comprising: (a) culturing a recombinant cell containing a vector comprising a recombinant nucleotide sequence as depicted in FIG. 3A (SEQ ID NO:9), such that the xcex11xe2x86x922fucosyltransferase protein, encoded by SEQ ID NO:9, is expressed by the cell; and (b) recovering the expressed protein or a cellular fraction containing said protein. In one embodiment, the invention provides the purified protein produced by the method. In another embodiment, the invention provides a cellular fraction with xcex11xe2x86x922fucosyltransferase protein activity.
This invention provides a method of producing a rat xcex11xe2x86x922fucosyltransferase protein comprising: (a) culturing a recombinant cell containing a vector comprising a recombinant nucleotide sequence encoding a protein sequence as depicted in FIG. 5 (SEQ ID NO:8), such that the xcex11xe2x86x922fucosyltransferase protein, encoded by SEQ ID NO:7, is expressed by the cell; and (b) recovering the expressed protein or a cellular fraction containing said protein. In one embodiment, the invention provides the purified protein produced by the method. In another embodiment, the invention provides a cellular fraction with xcex11xe2x86x922fucosyltransferase protein activity.
This invention provides a method of producing a rat xcex11xe2x86x922fucosyltransferase protein comprising: (a) culturing a recombinant cell containing a vector comprising a recombinant nucleotide sequence encoding a protein sequence as depicted in FIG. 3A (SEQ ID NO:10), such that the xcex11xe2x86x922fucosyltransferase protein, encoded by SEQ ID NO:9, is expressed by the cell; and (b) recovering the expressed protein or a cellular fraction containing said protein. In one embodiment, the invention provides the purified protein produced by the method. In another embodiment, the invention provides a cellular fraction with xcex11xe2x86x922fucosyltransferase protein activity.
This invention provides a method for detecting the onset of liver cancer comprising the detection of the expression of a nucleotide sequence as depicted in FIG. 5 (SEQ ID NO:9) or a fragment or complement thereof.
This invention provides a method to suppress or inhibit from a cell the function of the protein of the invention, which method comprises contacting said cell with a nucleic acid comprising a nucleotide sequence that is the reverse complement of a nucleotide sequence as depicted in FIG. 5 (SEQ ID NO:7) or a fragment thereof, or as depicted in FIG. 3A (SEQ ID NO:9) or a fragment thereof, and wherein when said nucleic acid is RNA, the base T (thymine) in SEQ ID NO:7 and SEQ ID NO:9 is substituted by the base U (uracil). In one embodiment, said nucleic is contained within an adenoviral or retroviral vector. In another embodiment, the cell is a human small cell lung carcinoma cell.
The invention provides methods for the preparative synthesis of compositions comprising Fucxcex11xe2x86x922Galxcex21xe2x86x923GalNAc, said methods comprising contacting isolated or purified rat xcex11xe2x86x922fucosyltransferase or a cellular fraction containing xcex11xe2x86x922fucosyltransferase with GDP-fucose and a molecule having a terminal Galxcex21xe2x86x923GalNAc moiety. The molecule having a terminal Galxcex21xe2x86x923GalNAc moiety can be a glycolipid, a glycoprotein, a glycolipoprotein or a free saccharide.
Thus, the invention provides methods for the preparative synthesis of glycolipids, glycoproteins, glycolipoproteins or free oligosaccharides comprising Fucxcex11xe2x86x922Galxcex21xe2x86x923GalNAc. In one embodiment, the fucosyl-glycolipid, -glycoprotein, -glycolipoprotein or -free oligosaccharide or -polysaccharide produced by the method of the invention is used as an additive to a nutritional formula.
In a particular embodiment, the invention provides a method for the preparative synthesis of fucosyl-GM1 comprising contacting isolated or purified rat xcex11xe2x86x922fucosyltransferase or a cellular fraction containing xcex11xe2x86x922fucosyltransferase with GDP-fucose and the ganglioside GM1 and recovering fucosyl-GM1.
The invention provides methods for the use of fucosyl-GM1 in immunotherapy for human disease comprising administering said compound to a human patient with a disease. In one embodiment, the disease is cancer or neurological disease. In a specific preferred embodiment, said patient has small cell lung carcinoma.
3.1. Abbreviations
As used herein, the following abbreviations shall have the meanings indicated.
AAF: N-2-acetylaminofluorine
xcex11xe2x86x922FucT: xcex11xe2x86x922fucosyltransferase
cDNA: complementary DNA
FucT, fucosyltransferase
fucosyl-GM1:II3NeuAcIV3FucGg4, Fucxcex11xe2x86x922Galxcex21xe2x86x923
GalNAcxcex21xe2x86x924[NeuAcxcex12xe2x86x923]Galxcex21xe2x86x924Glcxcex21xe2x86x921 Cer
ganglio-B: II3NeuAcIV3xcex1GalIV2FucGg4, Galxcex11xe2x86x923[Fucxcex11xe2x86x922]
Galxcex21xe2x86x923GalNAcxcex21xe2x86x924[NeuAcxcex12xe2x86x923]Galxcex21xe2x86x924Glcxcex21xe2x86x921Cer
GM1:II3NeuAcGg4, Galxcex21xe2x86x923GalNAcxcex21xe2x86x924[NeuAcxcex12xe2x86x923]Galxcex21xe2x86x924
Glcxcex21xe2x86x921Cer
nLc4: lactoneotetraosylceramide or
Galxcex21xe2x86x924GcNAcxcex21xe2x86x923Galxcex21xe2x86x924Glcxcex21xe2x86x921Cer
PCR: polymerase chain reaction
RT-PCR: reverse transcriptionxe2x80x94polymerase chain reaction
FIG. 1. Portions of aligned nucleotide sequences of human (SEQ ID NO.""s:12-20) and rabbit (SEQ ID NO.""s:21-29) xcex11xe2x86x922FucT nucleic acids. The regions corresponding to forward and reverse primers used in the Example described infra in Section 6 are indicated except for Primer III (SEQ ID NO:3) which corresponds to the most 3xe2x80x2 end of the open reading frame.
FIG. 2. RT-PCR analysis of rat hepatoma H35 cell total RNA. Lane 1, RT-PCR product generated using primers I (SEQ ID NO:1) and II (SEQ ID NO:2); lane 2, RT-PCR product generated using primers I (SEQ ID NO:1) and III (SEQ ID NO:3); lane 3, RT-PCR product generated using primers V (SEQ ID NO:5) and III (SEQ ID NO:3). Seven xcexcl of each PCR mix was electrophoresed in a 0.8% agarose gel in 1xc3x97 TBE buffer. The gel was stained with ethidium bromide. Size standards of 1.0, 0.75, and 0.5 kb are indicated.
FIGS. 3(A-B). Nucleotide (SEQ ID NO:9) and deduced amino acid sequence (SEQ ID NO:10) of the catalytic domain of rat hepatoma H35 cell xcex11xe2x86x922FucT. FIG. 3A.
Nucleotide and deduced amino acid sequence of the 1068-bp rat hepatoma H35 cell xcex11xe2x86x922 FucT RT-PCR product generated with primers V (SEQ ID NO:5) and III (SEQ ID NO:3). The sequence extends from the second C residue following the EcoRI site in primer V through the end of primer III (SEQ ID NO:3). This nucleotide sequence has been deposited in GenBank with the Accession No. AF042743. The sequence is translated in reading frame 1. Potential N-linked glycosylation sites are shaded. The region which overlaps rat FTB is indicated by a solid line over the sequence. The amino acid differing between the H35 cell sequence and that predicted by the rat FTB sequence is underlined. The stop codon is indicated in bold lettering. FIG. 3B. Comparison of amino acid sequence homology between the catalytic domain of rat hepatoma H35 cell xcex11xe2x86x922FucT and human Sec2 (SEQ ID NO:11).
FIG. 4. TLC analysis of reaction products from transfer of [14C]fucose to GM1 and nLc4 catalyzed by the pPROTA-expressed catalytic domain of rat hepatoma H35 cell xcex11xe2x86x922FucT. Lanes 1 and 3 show results from pPROTA expressed H35 cell xcex11xe2x86x922FucT in the forward orientation. Lanes 2 and 4 show results from pPROTA expressed H35 cell xcex11xe2x86x922FucT in the reverse orientation. Lanes 1 and 2, transfer to GM1; lanes 3 and 4,transfer to nLc4. The arrow indicates the TLC mobility of standard fucosyl-GM1. The solvent system was composed of CHCl3:CH3OH:H2O (60:40:9), containing 0.02% CaCl2.2H2O. See, infra, Section 6 for details.
FIG. 5. Nucleotide (SEQ ID NO:7) and deduced amino acid sequence (SEQ ID NO:8) of the 1140 bp rat hepatoma H35 cell xcex11xe2x86x922FucT RT-PCR product generated with primers VI (SEQ ID NO:6) and III (SEQ ID NO:3). The entire coding region of 380 amino acids through the stop codon is represented. Potential N-linked glycosylation sites are highlighted. The region which was found to overlap rat FTB is indicated by a solid line over the sequence. The amino acid differing between the H35 sequence and that predicted by the rat FTB is underlined. The intra-cellular/transmembrane domain comprised of 81 nucleotides (27 amino acids), is shown in larger italic font.
FIG. 6. TLC analysis of reaction products from transfer of [14C] to GM1 catalyzed by expressed recombinant full length rat hepatomxcex11xe2x86x922FucT. Lane A: transfer to GM1 in absence of detergent or phospholipid; Lane B: transfer to GM1 in the presence of phosphatidylglycerol (PPG), Lane C: transfer to GM1 in the presence of PPG and G3634A detergent, and Lane D: transfer to GM1 in the presence of CHAPSO detergent. The reactions were conducted for two hours at 37xc2x0 C. GM1 standard is indicated. The solvent system was composed of CHCl3:CH3OH:H2O (60:40:9), containing 0.02% CaCl2.2H2O. See, infra, Section 7 for details.
FIG. 7. PCR products generated using primers I (SEQ ID NO:1) and II (SEQ ID NO:2) in RT-PCR analysis total RNA from rat hepatoma H35 cells and from normal rat liver tissue. RT-PCR analysis was performed on Lane 1: Total RNA from rat hepatoma H35 cells, Lane 2: Total RNA from normal rat liver tissue, and Lane 3: Total RNA from AAF-fed rat liver tissue. The arrow on right indicates location of 0.6-kb PCR product. Size markers (in kb) are indicated on left. Five xcexcl of each PCR mix was electrophoresed in a 0.8% agarose gel in 1xc3x97 TBE buffer. The gel was stained with ethidium bromide. See, infra, Section 8 for details.
FIGS. 8(A-B). FIG. 8A. TLC Analysis of reaction products from transfer of [14C] to GM1 catalyzed by full length expressed recombinant xcex11xe2x86x922FucT from COS-7 cells transfected with FL-RFT-pcDNA3 in the presence of increasing concentrations of antisense FL-RFT(xe2x88x92)-pcDNA3. All reactions were carried out in the presence of CHAPSO detergent. Equimolar ratios of total DNA were maintained in each transfection by including varying concentrations of pcDNA3 plasmid (vector minus insert). All lanes except Lane II were transfected with 1 xcexcg of FL-RFT-pcDNA3. Total FL-RFT(xe2x88x92)-pcDNA3 transfected was as follows: Lane Ixe2x80x940 xcexcg, Lane IIxe2x80x941.0 xcexcg, Lane IIIxe2x80x941.0 xcexcg, Lane IVxe2x80x942.0 xcexcg, Lane Vxe2x80x943.0 xcexcg, and Lane VIxe2x80x945.0 xcexcg. The solvent system was composed of CHCl3:CH3 OH:H2O (60:40:9), containing 0.02% CaClH2.2H2O. The GM1 standard was visualized by spraying in 0.5% orcinol in 2 N sulfuric acid; FIG. 8B. Percentage reduction of initial xcex11xe2x86x922FucT activity by increasing doses of FL-RFT(xe2x88x92)-pcDNA3. The major reaction product in each lane (indicated by arrow) (see FIG. 8A) was scraped off the plate and counted in a scintillation counter. Cpm minus background counts of 117 (Lane II) and percentage reduction of initial xcex11xe2x86x922FucT activity by increasing doses of FL-RFT(xe2x88x92)-pcDNA3 are shown. See, infra, Section 9 for details.
FIG. 9. Preparative in vitro biosynthesis of fucosyl-GM1 utilizing recombinant rat xcex11xe2x86x922fucosyltransferase. The results demonstrate the appearance of increasing amounts of a slower migrating band corresponding to fucosyl-GM1 from transfer of fucose in the xcex11xe2x86x922-linkage to the added GM1 acceptor with time. The enzyme is very active, yielding almost complete conversion to fucosyl-GM1 after 24 to 48 hours. See, infra, Section 10 for details.